The present invention relates to a method for measuring metals in sample of living body and a reagent for the measurement. More closely, the present invention relates to a method for measuring metals in samples of living body, in which metals other than the objective metals of measurement are added to the measurement system to release the objective metals bonding to co-existing substances in the sample from the co-existing substances, so that even trace objective metals in the sample can be measured with high accuracy; and a reagent for the measurement, in which metals other than the objective metals of the measurement are contained in a reagent for measuring metals in a sample of living body in order to release the objective metals bonding to the co-existing substances in the said sample from the co-existing substances.
In the field of clinical examination, the measurement of various kinds of metals in samples (components) of living body has frequently been used as indexes of disease diagnosis. For example, the measurement of magnesium as a diagnostic means for ischemic heart disease, hyperthyroidism, etc.; the measurement of iron as a diagnostic means for various kinds of anemia, chronic hepatopathy, etc.; the measurement of copper as a diagnostic means for disease of hepatic duct, anemia, etc.; the measurement of calcium as a diagnostic means for nephropyelitis, nephrosis, etc.; and the measurement of zinc as a diagnosis means for disease of circulatory organs, hemolytic anemia, etc. have usually been used. Other metals in living body including aluminum, nickel, manganese, chromium and so on have been also measured as indexes of various kinds of diseases.
The typical methods of measurement of these metals are, for example, atomic absorption spectrometry, emission spectrochemical analysis, X-ray fluorescence analysis, voltammetry, chelatometry, and the like. However, these methods are inadequate, particularly for daily examinations in the clinical examination field, since they not only require special, expensive apparatuses but also pretreatments of samples in such methods are troublesome. Therefore, recently, other various kinds of methods for measuring metals in living body have been developed. Among these, the methods for measuring major metals are, for example, as follows:
(i) As the method for measuring magnesium, there have been known the colorimetry in which titan yellow and xylidyl blue are used as color-producing agents, the enzymatic method which utilizes the activation of the enzyme reaction which is one of the physiological actions of magnesium (Japanese Patent Application Laid-open Nos. 124398/1986 and 30597/1989), and the like. PA1 (ii) As the method for measuring iron, there have been frequently used the colorimetry in which bathophenanthroline and tripyridil-triazine are used as color-producing agents after reduction of free trivalent iron ions into bivalent iron ions with a reducing agent such as ascorbic acid and the like, and various kinds of other color-producing agents have been developed (Japanese Patent Application Laid-open Nos. 144750/1983 and 301678/1989). PA1 (iii) As the method for measuring copper, there has been known the colorimetries in which bathocuproinedisulfonic acid and the like are used as color-producing agents after addition of reducing agent, and in which co-existing metals are masked and the 2-pyridylazo-aminophenol derivatives are then used as the color-producing agents (Japanese Patent Application Laid-open No. 69552/1985), and so on. PA1 (iv) As the method for measuring calcium, there has been frequently used the colorimetry in which .sigma.-cresolphtalein complexone is used as a color-producing agent under alkaline condition. In addition, there has also been known the enzymatic method which utilizes the action of the enzyme activity as an activating factor which is one of the physiological actions of calcium (Japanese Patent Application Laid-open No. 142498/1990). PA1 (v) As the method for measuring zinc, there has been frequently used the colorimetry in which zinc is reacted with 2-(5-bromo-2-pyridyl)azo!-5-(diethylamino)phenol in the presence of surfactants to form a complex compound. PA1 (a) As mentioned above, when the substances able to bond to the objective metals of measurement (hereinafter, referred to as interfering chelating substance), which are derived from patients themselves or the treatment methods, exist in the samples before the detecting reaction of the objective metals being occurred, the objective metals may exist in a state where they have been already bonded to the interfering chelating substances in the sample. PA1 (b) In such sample, a sufficient reaction does not occur by addition of the reacting reagents of detection (e.g., color-producing substances) to the sample. Therefore, the colorimetry of the resulting color developments leads to inaccurate determined values, or the measurement itself sometimes become impossible. That is, such inaccuracy of measured values in the colorimetry, in which the color developments occurred by addition of the color-producing substances to the measurement system are detected, are caused by the information or hardness of formation of chelate complexes of the color-producing agents which develop color by specifically bonding to the objective metals. PA1 (c) In the methods applying the enzyme activities, the inaccuracy of the measured values are caused by the physiological action between the essential metals for expression of enzyme activity or the metals interfering with the enzyme activity and the enzyme is inhibited by the existence of the interfering chelating substances. In such case, the addition of metals other than the objective metals which do not inhibit or, if any, hardly inhibit the expression of enzyme activity in the detecting reaction system, makes it possible to measure the metals accurately in living body fluid. PA1 (a) magnesium: xylidyl blue, .delta.-hydroxy-5-quinolinesulfonic acid; PA1 (b) calcium: .sigma.-cresolphtalein complexone; PA1 (c) copper: bathocuproine, 2-(5-bromo-2-pyridyl)azo!-5-N-propyl-N-(3-sulfopropyl)amino!phenol, 2-(2-thiazolylazo)-5-N-ethyl-N-sulfopropylamino benzoic acid (TSAB); PA1 (d) zinc: 2-(5-bromo-2-pyridyl)azo!-5-(diethylamino)phenol (5-Br-PADAP); PA1 (e) iron: bathophenanthroline, tripyridil-triazine, ferrozine, .sigma.-phenanthroline, 2-nitroso-5-(N-propyl-N-sulfopropylamino)-phenol (Nitroso-PSAP); PA1 (f) aluminum: 1-(1-hydroxy-4-methyl-2-phenylazo)-2-naphtol-4-sulfonic acid; PA1 (g) cadmium: .alpha., .beta., .gamma., .delta.-tetraphenylporphine tetrasulfonate; PA1 (h) chromium: 3,3'-di(N-methyl-N-carboxymethylaminomethyl)-.sigma.-cresolsulfonphtalein; PA1 (i) mercury: diphenylthiocarbazone; PA1 (j) lead: diphenylthiocarbazone, 2,7-bis(4-methyl-2-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid; PA1 (k) nickel: 2-furildioxime; and PA1 (l) selenium: 3,3-diaminobendizine.
The tested samples of living body for measurement of metals are those of blood (whole blood, blood serum, blood plasma, blood cell), urine, feces, hair, saliva, breast milk, and so on. Generally speaking, in clinical examination, metals contained in blood or urine are mainly measured.
In the measurement of metals in sample of living body using the above-mentioned metal measuring methods, color-producing agents which specifically react (bond) with the objective metals (i.e., chelating agents) are generally used. In the above-mentioned enzymatic methods, the enzymes which are specifically acted on by the objective metals are chosen, and the existing amount of metals are determined quantitatively according to the variability of the enzyme activity values.
However, in the measurement using the enzymatic methods and the colorimetries which utilize the color-producing agents mentioned above, desirable data sometimes cannot be obtained depending on samples. That is, the phenomenon, where the formability of a complex compound between the objective metal of measurement and the color-producing agent or the physiological action of the objective metals against the enzyme is inhibited or interfered with by some factors, sometimes can be observed.
Various factors are considered as causes of such phenomena. Among them, one possibility can be assumed that they may be caused by insufficient expression of the objective detection reaction, in which the objective metals of measurement in sample bond to some kinds of co-existing substances or secondary added substances in living body fluid and, consequently, the objective metals are subjected to the measurement system as the bonding state is maintained.
There are two types to be considered as such samples, which contain the bonding substances derived from patients themselves, and which contain the bonding substances derived from the treatment methods carried out after sampling from the patients. For example, in the samples containing the bonding substances derived from patients themselves, the substances are those derived from patients who have been dosed pharmaceutical medicines containing components having a metal-bondability (e.g., eliminant of renal calculus, detoxifying agents, metal-eliminating agents, anti-coagulants, etc.). In the samples containing the bonding substances derived from the treatment methods, the substances are, for example, those derived from various kinds of treatment agents charged in a blood sampling tube. Embodiments of such treatment agents include various derivatives from ethylenediamine (e.g., ethylenediaminetetraaketates, etc.), deferoxamine mesilate, citric acid, tartaric acid, etc., which largely affect the detection of the objective metals particularly. These are just a part of embodiments of substances which can bond to the objective metals in living body fluid or sample, and many other substances can be included.
In the measurement of metals in such samples, the objective metals of measurement can not induce the desired reaction in the detection system, depending on the combination of the objective metals and the co-existing substances mentioned above. Accordingly, the quantitative relationship between the color-production amount or the enzyme activity and the existing amount of the objective metals may be lost. For example, it is well known that sodium ethylenediaminetetraacetate (hereinafter, referred to as EDTA) can react with almost all metallic ions, except monovalent metallic ions, to form stable, water-soluble chelate complexes. However, when EDTA or other substances having similar natures to EDTA are co-existing in samples, they may form chelate complexes with the objective metals of measurement before detecting a reaction being occurred out and, therefore, the accurate measurement of the objective metals would become impossible. Particularly, in cases where the substances bonding to the objective metals of measurement are derived from patients themselves, it is impossible to predict previously in the clinical examination scene whether the samples contain disadvantageous factors to measurement of the objective metals or not. Therefore, when the question about the measured values occurs, re-sampling and re-examination may be naturally carried out, which may lead to burdens not only to patients but also in the scene of clinical examination.
In view of the foregoing, the present inventors have carried out extensive studies to provide a method and a reagent for measuring metals in vital components, in which the objective metals can be measured in high accuracy even in the presence of various interfering substances which may greatly affect the measured values in the measurement of metals in samples of living body. As a result, the present invention has been completed.